In eukaryotes, the 5′ ends of most messenger RNAs (mRNAs) are blocked, or “capped.” In addition, there are some other forms of RNA that are also capped, for instance small nuclear RNAs (snRNAs). The cap contains a 5′-5′ triphosphate linkage between two nucleoside moieties and a 7-methyl group on a distal guanine ring. The capping of mRNA and snRNA promotes their normal functions in cells.
The ability to synthesize capped RNA molecules in vitro is useful, because it allows workers to prepare RNA molecules that behave properly in a variety of biological applications. Such applications include both research applications and commercial production of polypeptides, e.g., the production in a cell-free translation system of polypeptides containing an “unnatural” amino acid at a specific site, or production in cultured cells of polypeptides that require post-translational modification for their activity or stability. In the latter systems, synthesis proceeds for a considerably longer time and therefore produces more protein.
The method most frequently used to make capped RNAs in vitro is to transcribe a DNA template with either a bacterial or bacteriophage RNA polymerase in the presence of all four ribonucleoside triphosphates and a cap dinucleotide such as m7G(5′)ppp(5′)G (henceforth m7GpppG). The polymerase initiates transcription with a nucleophilic attack by the 3′-OH of the Guo moiety of m7GpppG on the α-phosphate of the next templated nucleoside triphosphate, resulting in the initial product m7GpppGpN. The alternative, GTP-initiated product pppGpN is suppressed by setting the ratio of m7GpppG to GTP between 5 and 10 in the transcription reaction mixture.
Synthetic RNAs may be synthesized by cell-free transcription of DNA templates. See R. Contreras et al., “Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes,” Nucl. Acids Res., vol. 10, pp. 6353-6362 (1982); J. Yisraeli et al., “Synthesis of long, capped transcripts in vitro by SP6 and T7 RNA polymerases, pp. 42-50 in J. Dahlberg et al. (Eds.), Meth. Enzymol., vol. 180., pp. 42-50 (1989); and D. Melton et al., “Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter,” Nucl. Acids Res., vol. 12, pp. 7035-7056 (1984).
Capped RNAs thus produced are active in splicing reactions carried out in vitro. See M. Konarska et al., “Recognition of cap structure in splicing in vitro of mRNA precursors. Cell, vol. 38, pp. 731-736 (1984); and I. Edery et al., “Cap-dependent RNA splicing in a HeLa nuclear extract,” Proc. Natl. Acad. Sci. USA, vol. 82, pp. 7590-7594 (1985).
Capped mRNAs are translated in cell-free translation systems more efficiently than are non-capped mRNAs. See S. Muthukrishnan et al., “5′-Terminal 7-methylguanosine in eukaryotic mRNA is required for translation,” Nature, vol. 255, pp. 33-37 (1975); L. Chu et al., “Paradoxical observations on the 5′ terminus of ovalbumin messenger ribonucleic acid,” J. Biol. Chem., vol. 253, pp. 5228-5231 (1978); E. Darzynkiewicz et al., β-Globin mRNAs capped with m7G, m22.7G or M32.2.7G differ in intrinsic translation efficiency,” Nucl. Acids Res., vol. 16, pp. 8953-8962 (1988); and E. Darzynkiewicz et al., “Inhibition of eukaryotic translation by nucleoside 5′-monophosphate analogues of mRNA 5′-cap: Changes in N7 substituent affect analogue activity,” Biochem., vol. 28, pp. 4771-4778 (1989).
5′-Unmethylated mRNAs are translationally less active than 5′-methylated mRNAs. See G. Both et al., “Methylation-dependent translation of viral messenger RNAs in vitro,” Proc. Natl. Acad. Sci. USA, vol. 72, pp. 1189-1193 (1975).
Capped mRNAs introduced into cultured mammalian cells by electroporation are translated more efficiently than are non-capped mRNAs. See E. Grudzien et al., “Differential inhibition of mRNA degradation pathways by novel cap analogs,” J. Biol. Chem. vol. 281, pp. 1857-1867 (2006).
A. Pasquinelli et al., “Reverse 5′ caps in RNAs made in vitro by phage RNA polymerases,” RNA, vol. 1, pp. 957-967 (1995), reported that bacteriophage polymerases use the 3′-OH of the 7-methylguanosine moiety of m7GpppG to initiate transcription, demonstrating that approximately one-third to one-half of RNA products made with this cap analogue actually contain the cap in reversed orientation, i.e., Gpppm7GpN. Such reverse-capped RNA molecules behave abnormally. The same authors reported that when reverse-capped pre-U1 snRNA transcripts were injected into Xenopus laevis nuclei, they were exported more slowly than natural transcripts. Similarly, cytoplasmic reverse-capped U1 snRNAs in the cytoplasm were not properly imported into the nucleus.
The presence of a cap on mRNA strongly stimulates translation of an mRNA transcript into protein. E. Grudzien et al., “Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency,” RNA vol. 10, pp. 1479-1487 (2004), demonstrated that mRNAs containing caps incorporated exclusively in the reverse orientation were translated in a cell-free system with only 4% the efficiency of mRNAs containing caps incorporated exclusively in the normal orientation.
J. Stepinski et al., “Synthesis and properties of mRNAs containing the novel ‘anti-reverse’ cap analogues 7-methyl(3′-O-methyl)GpppG and 7-methyl(3′-deoxy)GpppG,” RNA, vol. 7, pp. 1486-1495 (2001) reported the synthesis and use of two novel two novel cap analogs, m73′ dGpppG and m27,3′-OGpppG, that are incapable of being incorporated in the reverse orientation. mRNAs capped with these “anti-reverse cap analogs” (ARCAs) were translated more efficiently in an in vitro system than mRNAs capped with the conventional analog, m7GpppG. See also U.S. Pat. No. 7,074,596, and U.S. Patent Application Publication 2003/0194759.
Z. Peng et al., “Synthesis and application of a chain-terminating dinucleotide mRNA cap analog,” Org. Lett., vol. 4, pp. 161-164 (2002) reported the synthesis of m27,3′-OGpppG and its use in the in vitro transcription of homogeneously capped RNA.
J. Jemielity et al. “Novel ‘anti-reverse’ cap analogues with superior translational properties,” RNA, vol. 9, pp. 1108-1122 (2003) reported that substitution at the 2′ position with either —OCH3 or —H, to produce m27,2′-OGpppG or m72′ dGpppG, respectively, yielded ARCAs with properties equivalent to or slightly more favorable than those of ARCAs substituted at the 3′ position as measured by the criteria of binding to the translational cap-binding protein eIF4E, correct incorporation into mRNA during in vitro transcription, and translational efficiency of the resulting mRNAs in a cell-free system.
The amount of protein produced from synthetic mRNAs introduced into cultured mammalian cells is limited by the degradation of mRNA by natural turnover processes. A major in vivo pathway of mRNA degradation is initiated by removal of the cap from intact mRNA by a specific pyrophosphatase, Dcp1/Dcp2, that cleaves between the α and β phosphates. E. Grudzien et al. “Differential inhibition of mRNA degradation pathways by novel cap analogs,” J. Biol. Chem., vol. 281, pp. 1857-1867 (2006) designed and synthesized a cap analog in which a methylene group replaced the O atom between α and β phosphate groups, m27,3′-OGppCH2pG, mRNAs capped with this analog were resistant to hydrolysis by recombinant human Dcp2 in vitro. When introduced into cultured cells, mRNAs capped with m27,3′-OGppCH2pG were more stable than those capped with m27,3′-OGpppG.
There are two known decapping enzymes: Dcp1/Dcp2, which acts on intact mRNA to initiate 5′→3′ degradation; and DcpS, which acts on short capped oligonucleotides resulting from 3′→5′ degradation. Because Dcp1/Dcp2 or Dcp2 alone releases m7GDP from capped mRNAs, cleavage is likely to occur between the α- and β-phosphates. See Z. Wang et al., “The hDcp2 protein is a mammalian mRNA decapping enzyme,” Proc. Natl. Acad. Sci. U.S.A., vol. 99, pp. 12663-12668 (2002). Previously, it was shown that nucleoside 5′-monophosphorothioates as well as triphosphate analogs such as ATPγS, GTPγS, and GDPβS were stable towards phosphatases. See F. Eckstein et al., “Guanosine 5′-O-(2-thiodiphosphate). An inhibitor of adenylate cyclase stimulation by guanine nucleotides and fluoride ions”, J. Biol. Chem., vol. 254, pp. 9829-9834 (1979), and D. Cassel et al., “Activation of turkey erythrocyte adenylate cyclase and blocking of the catecholamine-stimulated GTPase by guanosine 5′-(gamma-thio) triphosphate”, Biochem Biophys Res Commun, vol. 77, pp. 868-873 (1977). Additionally, polynucleotides containing phosphorothioate internucleotide linkages were found to be degraded more slowly than their natural counterparts. See H. Matzura et al., “A polyribonucleotide containing alternation P═0 and P═S linkages”, Eur. J. Biochem., vol. 3, pp. 448-452 (1968). Interestingly, the diastereomers of phosphorothioates can exhibit different sensitivities toward nucleases. Nuclease P1 hydrolyses the Sp diastereomer more rapidly than the Rp. See B. Potter et al., “Synthesis and configurational analysis of a dinucleoside phosphate isotopically chiral at phosphorus. Stereochemical course of Penicillium citrum nuclease P1 reaction.”, Biochemistry, vol. 22, pp. 1369-1377 (1983). Ribonuclease T1 and snake venom phosphodiesterase preferably cleave the Rp diastereomer over the Sp. See F. Eckstein et al., “Stereochemistry of the transesterification step of ribonuclease T 1”, Biochemistry, vol. 11, pp. 3507-3512 (1972), and P. Burgers et al., “Absolute Configuration of the Diastereomers of Adenosine 5′-O-(1-thiotriphosphate): Consequences for the stereochemistry of polymerization by DNA-dependent RNA polymerase from Escherichia coli”, Proc. Natl. Acad. Sci. U.S.A., vol. 75, pp. 4798-4800 (1978).
Although mRNA capped with m27,3′-OGppCH2pG was more stable in cultured cells, it had lower translational efficiency, presumably because m27,3′-OGppCH2pG bound to eIF4E in vitro with considerably lower affinity than m27,3′-OGpppG. Thus, even though it was more stable in cultured cells, this advantage was offset by lower translational efficiency.
J. Kowalska et al. “Synthesis and properties of mRNA cap analogs containing phosphorothioate moiety in 5′,5′-triphosphate chain,” Nucleos. Nucleot. Nucl. Acids, vol. 24, pp. 595-600 (2005) reported synthesis of three cap analogs in which S is substituted for O in either the α, β, or γ phosphate moieties, e.g., m7GpsppG, m7GpppsG and m7GppS-CH3pG. These synthesized phosphorothioate cap analogs were more stable inhibitors of cap-dependent translation, and were resistant to DcpS decapping enzyme. However, these compounds would not show higher translational efficiency neither in vitro nor in vivo than regular ARCAs, because they would be incorporated to a large extent in the reverse orientation.
There is a need for a modification that would achieve both higher translation efficiency and increase resistance to both in vivo and in vitro degradation. The unique compounds reported here do both.